Ion channels underlie physiological functions from nerve excitation to blood-cell volume control as well as pathological conditions such as cystic fibrosis and lupus erythromatosis. Their structures and functions are not well understood in molecular terms. Only two of more than 50 types of channels, the acetylcholine receptor and the Na channel, have been modeled based on their nucleotide sequence. We plan to combine modern biophysical methods (patch clamp) with molecular-biological methods (recombinant DNA) to study ion channels native or foreign to the yeast, Saccharomyces cerevisiae. Our long-term goals are to understand the physiological roles of the native channels, to understand the control of channel synthesis and activities, and to study the fine-structure-fine-function relation of ion channels in general. The activities of the three yeast ion channels discovered in preliminary studies will be analyzed further with the patch clamp to study the channel kinetics, ion specificities and gating mechanisms. The possibility that some channels are inductible or repressible will be tested in cells grown in different conditions. The ion-dependent, ion-sensitive or drug-resistant mutants already isolated will be studied further genetically and electrically. Putative K+-channel mutants and osmotic-sensitive mutants will be isolated. When a mutant with a clear electric defect is identified, we will clone the channel gene, determine copy number, model and map the domains of the channel protein. We can then extend the study to second-site suppressors and find structure-function correlation. In collaboration with Patrick's laboratory, we will continue the cloning of the subunit genes of the mouse acetylcholine receptor into yeast by subcloning into shuttle bectors, transformation and mating to form a diploid harboring all four subunit genes. The expression of the acetylcholine receptor will be monitored with patch-clamp electrodes and by subaryldicholine-induced death. If the receptor is functionally expressed, we will create mutations at sites on the amphipathic chains and the large cytoplasmic domain and examine the unit-current behavior of the mutants. In collaboration with Mandel's and Davidson's laboratories, we will first probe the yeast genome for a possible native Na channel gene with Southern blots. Depending on the results, we plan either to study that native channel or to clone the vertebrate Na channel into yeast and study its structure-function relation through site-directed mutagenesis.